LOCUS       sequence-490798-        4991 bp    DNA     circular SYN 27-OCT-2025
DEFINITION  Bacterial expression of 2TMbeta protein under the pJex promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4991)
  AUTHORS   Doherty EE, Adler BA, Yoon PH, Hsieh K, Loi K, Armbuster EG, Lahiri 
            A, Bolling CS, Wilcox XE, Akkati A, Iavarone AT, Pogliano J, Doudna 
            JA
  TITLE     A miniature CRISPR-Cas10 enzyme confers immunity by an inverse 
            signaling pathway.
  JOURNAL   bioRxiv [Preprint]. 2025 Apr 1:2025.03.28.646030. doi: 
            10.1101/2025.03.28.646030.
  PUBMED    40196485
REFERENCE   2  (bases 1 to 4991)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 27, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Apr 1:2025.03.28.646030. doi: 
            10.1101/2025.03.28.646030."
COMMENT     Sequence Label: pBA1540
FEATURES             Location/Qualifiers
     source          1..4991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      253..475
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(1018..1833)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1018..1833)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1741..1760
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1825..1844
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     RBS             2602..2610
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      3541..3612
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3628..3655
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(4246..4991,1..205))
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg
       61 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
      121 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
      181 ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt taatcactat
      241 accaattgag atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta
      301 tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctata ccctctgtaa
      361 attccgctag acctttgtgt gttttttttg tttatattca agtggttata atttatagaa
      421 taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac
      481 tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa
      541 acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga
      601 atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag
      661 ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta
      721 tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg
      781 cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg
      841 ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc
      901 taatgcaccc agtaaggcag cggtatcatc aacaggctta cccgtcttac tgtccctgtg
      961 tcagatttct tactttattc aggcgattac gtgttacaac caattaacca attctgatta
     1021 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1081 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1141 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1201 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1261 atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc
     1321 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1381 ctgagcgagg cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1441 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1501 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1561 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     1621 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     1681 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     1741 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     1801 ggagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     1861 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     1921 attttgagac acaacgtggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca
     1981 gcacctgccc gaaccagaga cttatctcag gacgtcttac gaaaataact caagctgaat
     2041 aacgtgctgc agatggcggg tgaatgcggc atcgtctacc tccggcattc ccagcacgta
     2101 aatcccgtcc agtccacaga ccagcgaaat cagccgccag gcgatatttt cggcgctatc
     2161 gcgcagggta aattcgccag cggcatggcc cgcgcgaatg atcctgaccg cttcgtcatg
     2221 ccacaggttc atggtcagca ggtacgcgct tttgatttcc ggatcgctgt cggccagcag
     2281 ctgcgcctgg cgccacaggc ggatataggg ctccagccgt ccgtcctcgc tgccgagggc
     2341 ggaaaacagc tgttcacgcc agccggcggt gcgcgaaagg cgctgcagat ccatcatctc
     2401 gcggatcacg cggataaacg cctgggattt tagctcgccg gaggaggtga aatggtggtg
     2461 gacctggcct gcggcgacgc ccgcggcggt ggcgatgttg cggaccgtca taccggtaaa
     2521 cccttgatcc agcgcgacgc gcatggcggc ctgcatgatg gtttcccgac gttcttcgcg
     2581 attcagatag cccatattgt cacctcctta tccacacatt atacgagccg gatgattaat
     2641 ggggaacctt accagagggc gccccagctg gcaattcctc ttaagtaagt aagagtatac
     2701 gtatatcggc taataacgta ttaaggcgct tcggcgcctt tttttatggg ggtattttca
     2761 tcccaatcca cacgtccaac gcacagcaaa caccacgtcg accctatcag ctgcgtgctt
     2821 tctatgagtc gttgctgcat aacaaaaaac tgcaaaaaat agtttgacag gacacgtgtc
     2881 caactttgaa ttcaaaagat cttttaagaa ggagatatac tgatgtttag aataataaga
     2941 tttgactggt tgttaaaaac ggtatcacta atctctttgt ttgtcacaat actttcattt
     3001 tggatcttta agacagttct gtcttctgac ttaagcctgc tcaaattgtt atcaatatca
     3061 tcattagcat ccattatcat aaacttcttt ctcctttcat caaagatatc aagactaata
     3121 tggggaacaa taaggttttt caagaaagac atatatcctg acttaaatgg aatttgggtt
     3181 ggacatataa tcacagaaac aaatgatgaa tttgaagtcc gcgcaaaaat aaagcaagca
     3241 ctccttgtaa caaagataga aatgcacggc cccacagtta aatctgttac gctagaagca
     3301 acgccaacta aggaactgga taacaacaaa ttgtattatg tatataaatc aacccctaaa
     3361 aaccctagtt ggagcgagta tattggctcg actatattcg acgtaattga aagtaacaat
     3421 gcgctccaac tttccggtcg atattataca gacagaaaat ccgtggggag aatatcaata
     3481 aaaagaatat cacttaacac tgactccgat atatcttttt attaaaggat ctccaggcat
     3541 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     3601 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     3661 agaagaccaa ggaggacttt ggcaggtgct ctcctgagta ggacaaatcc gccgccctag
     3721 acctagggta cgggttttgc tgcccgcaaa cgggctgttc tggtgttgct agtttgttat
     3781 cagaatcgca gatccggctt cagccggttt gccggctgaa agcactattt cttccagaat
     3841 tgccatgatt ttttccccac gggaggcgtc actggctccc gtgttgtcgg cagctttgat
     3901 tcgataagca gcatcgcctg tttcaggctg tctatgtgtg actgttgagc tgtaacaagt
     3961 tgtctcaggt gttcaatttc atgttctagt tgctttgttt tactggtttc acctgttcta
     4021 ttaggtgtta catgctgttc atctgttaca ttgtcgatct gttcatggtg aacagctttg
     4081 aatgcaccaa aaactcgtaa aagctctgat gtatctatct tttttacacc gttttcatct
     4141 gtgcatatgg acagttttcc ctttgatatg taacggtgaa cagttgttct acttttgttt
     4201 gttagtcttg atgcttcact gatagataca agagccataa gaacctcaga tccttccgta
     4261 tttagccagt atgttctcta gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc
     4321 attgagatca tacttacttt gcatgtcact caaaaatttt gcctcaaaac tggtgagctg
     4381 aatttttgca gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc
     4441 tgatgtaatg gttgttggta ttttgtcacc attcattttt atctggttgt tctcaagttc
     4501 ggttacgaga tccatttgtc tatctagttc aacttggaaa atcaacgtat cagtcgggcg
     4561 gcctcgctta tcaaccacca atttcatatt gctgtaagtg tttaaatctt tacttattgg
     4621 tttcaaaacc cattggttaa gccttttaaa ctcatggtag ttattttcaa gcattaacat
     4681 gaacttaaat tcatcaaggc taatctctat atttgccttg tgagttttct tttgtgttag
     4741 ttcttttaat aaccactcat aaatcctcat agagtatttg ttttcaaaag acttaacatg
     4801 ttccagatta tattttatga atttttttaa ctggaaaaga taaggcaata tctcttcact
     4861 aaaaactaat tctaattttt cgcttgagaa cttggcatag tttgtccact ggaaaatctc
     4921 aaagccttta accaaaggat tcctgatttc cacagttctc gtcatcagct ctctggttgc
     4981 tttagctaat a
//
